Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC, respectively) are important agents of diarrheal disease that induce actin pedestals on intestinal epithelial cells beneath sites of bacterial attachment. They do so by injecting the host cell with proteins that ultimately activate a host cell regulator of actin assembly known as N-WASP. One critical effector for both pathogens is Tir, a bacterial protein that is inserted into the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. Despite these similarities, pedestal formation by EHEC and EPEC involve fundamentally different mechanisms. For EPEC, Tir is the only bacterial protein delivered to host cells that is required to induce formation of pedestals. Clustering of this protein in the host cell membrane promotes binding to Nck, a host adaptor protein that in turn activates N-WASP. In contrast, the Tir of EHEC neither binds to nor requires Nck for pedestal formation, and is not the only translocated bacterial protein required for pedestal formation by EHEC. Instead, EHEC requires a second translocated bacterial protein, termed EspFU, recently identified by our laboratory. Our current data support a model in which EspFU interacts directly with N-WASP to promote the formation of Tir/N-WASP complexes. However, unlike Nck, EspFU does not appear to interact directly with Tir. This observation implies that an unidentified host factor is likely to be required for the ultimate formation of Tir/N-WASP complexes. We propose to test and refine this model by: (1) delineating the elements of the cytoplasmic domain of EHEC Tir essential for actin assembly;(2) defining the essential interactions between Tir, EspFU and N-WASP, and identifying the putative host protein that is required for Tir-EspFU interaction, should we confirm its existence;(3) recapitulating Tir/EspFU-mediated actin assembly in cell-free extracts;(4) assessing the role of actin pedestal formation in intestinal colonization and the induction of tissue damage during EHEC infection.